Breast cancer is one of the most common malignancies in the United States, affecting one in nine women. The molecular mechanisms underlying the progression of breast cancer to more malignant behavior are not completely understood at the present time and are believed to involve deregulation of signaling pathways activated by growth factors and their rece!btors. For example, overexpression or overactivation of the human epidermal growth factor receptors by EGF family of ligands is frequently associated with an aggressive clinical course, decreased disease-free survival, and metastasis in human breast cancer. Despite the remarkable growth of information about growth factors and its receptors, the progress in understanding the mechanism by which downstream target(s) of these cell surface-initiated pathways regulate breast cancer progression remains elusive. As described below, our recent work indicate that growth factor-stimulation of p21-activated kinase (Pak1), a serine/threonine kinase, plays a significant role in the cell motility, invasiveness and survival, all of which are required for both normal mammary gland development and tumor formation. Our preliminary studies have discovered for the first time that PIN is a physiological substrate of Pak1 and of growth factor signaling and that Pak1-PIN pathway may be closely involved in breast cancer progression to more invasive phenotypes. Here we propose to investigate the molecular mechanism by which PIN participates in the development of enhanced cell survival, deregulated G1-S transition and tumorigenic phenotypes in breast cancer cells. Recent studies suggest that dynein light chain-8 (DLCS)/Protein Inhibitor of NOS (PIN) is a novel Pak1-intercating protein, growth factors and Pakl promotes interaction of endogenous Pakl and PIN, PIN is a physiologic substrate of Pak1 and its upstream activators such as heregulin and EGF, PIN is upregulated in MCF10AT progression model, and in mammary tumors from MMTVHER2 mice and human, PIN overexpression promotes growth-stimulation, anchorage-independence, and cell cycle progression, and Pak1 signaling is required and sufficient for PIN expression, and Pak1 signaling also regulates the expression of its substrate. Our working hypothesis is that "deregulation of Pak1 activity stimulates the expression and phosphorylation of PIN, and consequently, contributes to an enhanced cell survival, cell cycle progression and anchorage-independence, and tumorigenesis of breast tumor cells; these phenotypic effects of PIN might be controlled by Ser 88 phosphorylation of PIN by Pak1 ." To address these hypotheses, our specific aims are to determine: (1) The functional significance of Pak1-PIN interaction and the mechanism of Pak1 regulation of PIN expression in breast cancer cells; (2) The influence of PIN and its Ser 88 phosphorylation on the phenotypic changes associated with the progression of breast cancer cells; (3) The influence of PIN transgene expression on the biology, development and the early stages of tumorigenesis in mammary gland; and (4) The expression characteristics and significance of PIN and Pak1 during multi-step pathogenesis of breast carcinoma and in-patients with invasive breast cancer. These studies will uniquely define the mechanisms through which PIN and its upstream Pak1 kinase regulate survival, mitogenesis and tumorigenesis of breast cancer cells. Our proposed research is significant as this research might form the basis for new advances in identifying novel molecular targets, detecting, preventing and treating breast cancer, by identifying PIN as a key regulatory nodule.